Abstract
BackgroundSeveral lines of evidence suggest that CCL2 could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and facilitating nociceptive transmission in the spinal dorsal horn. The cellular and molecular mechanisms by which CCL2 enhances spinal pain transmission and causes hyperalgesia remain unknown. The substantia gelatinosa (lamina II) of the spinal dorsal horn plays a critical role in nociceptive transmission. An activated spinal microglia, which is believed to release pro-inflammatory cytokines including TNF-α, plays an important role in the development of neuropathic pain, and CCL2 is a key mediator for spinal microglia activation. In the present study, we tested the hypothesis that spinal CCL2 causes the central sensitization of substantia gelatinosa neurons and enhances spinal nociceptive transmission by activating the spinal microglia and augmenting glutamatergic transmission in lamina II neurons.MethodsCCL2 was intrathecally administered to 2-month-old male rats. An intrathecal injection of CCL2 induced heat hyperalgesia, which was assessed using the hot plate test. Whole-cell voltage-clamp recordings substantia gelatinosa neurons in spinal cord slices were performed to record glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs).ResultsThe hot plate test showed that 1 day after the intrathecal injection of CCL2 (1 μg), the latency of hind-paw withdrawal caused by a heat stimulus was significantly reduced in rats. One day after the intrathecal administration of CCL2, the amplitude of the evoked glutamatergic EPSCs and the frequency of spontaneous glutamatergic miniature EPSCs (mEPSCs) were significantly increased in outer lamina II neurons. Intrathecal co-injection of minocycline, a specific inhibitor of microglial activation, and CCL2 blocked the CCL2-induced reduction in the latency of hind-paw withdrawal and thermal hyperalgesia. Following intrathecal co-administration of CCL2 and minocycline, CCL2 failed to increase the frequency of glutamatergic mEPSCs and failed to promote glutamine release in lamina II neurons. Intrathecal co-injection of WP9QY, a selective TNF-α antagonist, and CCL2 completely inhibited CCL2-induced heat hyperalgesia and inhibited the increase in the frequency of glutamatergic mEPSCs in substantia gelatinosa neurons.ConclusionIn summary, our results suggest that an intrathecal injection of CCL2 causes thermal hyperalgesia by augmenting the excitatory glutamatergic transmission in substantia gelatinosa neurons through a presynaptic mechanism and facilitating nociceptive transmission in the spinal dorsal horn. Further studies show that intrathecal co-administration of minocycline, a specific inhibitor of microglial activation, or WP9QY, a selective TNF-α antagonist, completely inhibited CCL2 potentiation of glutamatergic transmission in substantia gelatinosa neurons and CCL2-induced heat hyperalgesia. The results of the present study suggest that peripheral nerve injury-induced upregulation of the spinal CCL2 level causes the central sensitization of substantia gelatinosa neurons by activating spinal microglia and that TNF-α mediates CCL2-induced thermal hyperalgesia and augmentation of glutamatergic transmission in lamina II neurons.
Highlights
Several lines of evidence suggest that CC chemokine ligand 2 (CCL2) could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and facilitating nociceptive transmission in the spinal dorsal horn
CCL2 is constitutively present in dorsal root ganglion (DRG) neurons with small or medium diameters and their processes in the dorsal horn of the spinal cord (DHSC), in which it is colocalized with transient receptor potential vanilloid receptor 1 (TRPV1) and the pain-related peptide, substance P (SP), or calcitonin gene-related peptide (CGRP) [2]
It is possible that CCL2 enhances glutamatergic transmission in substantia gelatinosa neurons by activating the spinal microglia. Consistent with this hypothesis, following intrathecal co-administration of CCL2 (1 μg) and minocycline (100 μg), CCL2 failed to increase the frequency of glutamatergic Miniature excitatory postsynaptic currents (EPSCs) (mEPSC) and failed to promote glutamine release by outer lamina II neurons (Figure 3B,C)
Summary
Several lines of evidence suggest that CCL2 could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and facilitating nociceptive transmission in the spinal dorsal horn. The presence of CCL2 in primary nociceptive afferent fibers and in secretory vesicles in the DHSC suggests that CCL2 is transported and subsequently released from the central terminals of nociceptive DRG neurons [2,5,8] In accord with this hypothesis, high potassium or capsaicin levels evoke a calcium-dependent secretion of CCL2 from DHSC explants of rats [2,5]. An intrathecal administration of antiCCL2 antibodies inhibits neuropathic pain behavior [12] These findings suggest that CCL2 could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and enhancing nociceptive transmission in the spinal dorsal horn. Further study is required to investigate the cellular and molecular mechanisms by which CCL2 facilitates pain transmission in the spinal dorsal horn
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